ML, AK, KB, and PK have ownership in Epic Sciences

ML, AK, KB, and PK have ownership in Epic Sciences.. 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAb) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification (WGA) and copy number variation (CNV) analysis. Results Based on CSPG4 expression and nuclear size, 1 to 250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5 to 371.5 CMCs/ml). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this population may contribute to develop effective therapeutic strategies. axis) across genomic regions (axis); e and f, candidate genes located in the amplified and deleted genomic regions. PMBCs (), excluded candidate cells () (see supplementary figure S2) and cells displayed in detail in c and d (). Novel chromosomal amplifications (*) (see supplementary table S1). CMC levels and clinical outcome of melanoma patients The number of patients in this study (n = 40) was not powered for survival analysis nor was the sampling of blood controlled for a specific line of therapy. Nevertheless, there was an association between the number of CMC per ml of blood and the short survival observed in some patients (table 2). A receiver operating characteristic (ROC) curve was constructed using the clinical outcome from melanoma patients (n = 39). Alvimopan (ADL 8-2698) A cutoff value of 8.7 CMC/ml was determined from this cohort data. The mean overall survival time for patients with 8.7 CMC/ml which was 315.9 days was significantly longer than that for those with 8.7 CMC/ml, which was 18 days. Discussion Given the implementation of emerging targeted therapies for metastatic melanoma in the clinic, a complete evaluation of the overall genomic tumor heterogeneity in individual patients using captured CMCs could provide better therapeutic directions than that from a single biopsy (35C37). Technical progress in the field of CTC has led to Rabbit Polyclonal to ANXA1 the development of several methodologies that have enabled cellular detection Alvimopan (ADL 8-2698) and characterization of CTCs. Although several groups including ours have provided convincing lines of evidence of the biological Alvimopan (ADL 8-2698) and clinical significance of CTCs with respect to epithelial cancers (22,38), similar approaches for melanoma seem to be inadequate due to the low recovery rate observed. For example, Rao and, more recently Khoja evaluated the CellSearch? system using CD146 coated immunomagnetic beads for CMC isolation in 44 and 101 advanced melanoma patients, respectively. Using a threshold of 2 CMCs per 7.5 ml of blood, they reported positive results in about 25% of the patients analyzed (13,14). Whether the low frequency of CMCs in comparison to CTCs (28) is due to the biology of melanoma and/ or to the low sensitivity of the currently available methods and biomarkers is unknown. We have demonstrated that the HDSCA using a CSPG4 antibody cocktail has successfully identified CMCs in 55% of patients with advanced melanoma using a threshold of 1 1 CMCs per test (0.2-2ml of blood). Due to the highly heterogeneous expression of candidate protein markers in clinical diagnosis of primary and metastatic melanomas (39), several distinct markers such as Tyrosinase, MAGE-3, MART-1, CD146 have been proposed for CMC detection (40C42). In this study, we elected to target CSPG4. The use of seven mAbs against different and spatially distant epitopes of this proteoglycan identified CSPG4 expression in all melanoma cell lines and yielded detection of 97% of those cells independently of their expression level (Figure 1). The robust dynamic range of the CSPG4 assay was also demonstrated in the high-count melanoma cases #30 and #37 where we detected cells with very low CSPG4 expression and showed by HMB-45 characterization and CNV profiling that they belonged to the cancer lineage. To extend the discrimination of the CMC.